基于高通量测序技术研究靛玉红对溃疡性结肠炎小鼠肠道菌群的影响  

Effect of indirubin on intestinal flora in mice with ulcerative colitis based on high-throughput sequencing technology

作　　者：梁艳妮[1] 程雯 吴柯楠 于金高 张东博[1] 张珍[1] 王征[1] LIANG Yan-ni;CHENG Wen;WU Ke-nan;YU Jin-gao;ZHANG Dong-bo;ZHANG Zhen;WANG Zheng(Co-construction Collaborative Innovation Center for Chinese Medicine Resources Industrialization by Shaanxi&EducationMinistry,State Key Laboratory of Research&Development of Characteristic Qin Medicine Resources(Cultivation),ShaanxiInnovative Drug Research Center,Shaanxi University of Chinese Medicine,Xianyang 712083,China)

机构地区：[1]陕西中医药大学陕西中药资源产业化省部共建协同创新中心,秦药特色资源研究开发国家重点实验室(培育),陕西省创新药物研究中心,陕西咸阳712083

出　　处：《中草药》2021年第13期3896-3904,共9页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81803951);国家自然科学基金资助项目(81973687);陕西省教育厅重点科研项目(20JY012);陕西高校青年创新团队(陕教[2019]90号);陕西高校第三批“青年杰出人才支持计划”项目(陕教工[2019]95号);陕西省首批中医药优秀中青年科技骨干人才支持计划。

摘　　要：目的研究靛玉红对葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的溃疡性结肠炎(ulcerative colitis,UC)模型小鼠肠道菌群的影响及其作用机制。方法雄性昆明种小鼠随机分为对照组、模型组、柳氮磺胺吡啶(125 mg/kg)组和靛玉红低、中、高剂量(7.5、15.0、30.0 mg/kg)组,采用3%DSS自由饮用7 d诱导小鼠UC模型,第8天ig药物,连续干预7 d后,观察小鼠体征进行疾病活动指数(disease activity index,DAI)评分;采用苏木素-伊红(HE)染色观察小鼠结肠组织病理变化;采用ELISA法检测小鼠血清中白细胞介素-6(interleukin-6,IL-6)、IL-8、IL-10、IL-1β和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;各组收集5份小鼠肠道内容物样本,采用细菌16S rRNA基因V4~V5区高通量测序,分析小鼠肠道内容物的菌群变化。结果与模型组比较,靛玉红组小鼠DAI评分显著降低(P<0.05),结肠组织损伤减轻,炎性细胞浸润减少,血清中促炎细胞因子IL-6、IL-8、IL-1β和TNF-α水平显著降低(P<0.05),抗炎细胞因子IL-10水平呈升高趋势。16S rRNA基因测序结果显示,模型组小鼠肠道菌群多样性降低,靛玉红组小鼠肠道菌群多样性升高,在门水平主要表现为拟杆菌门与厚壁菌门比例回升,属水平7个菌属Lachnospiraceae_FCS020_group、Ruminiclostridium_9、Eubacterium_brachy_group、别样棒菌属Allobaculum、链球菌属Streptococcus、Erysipelotrichaceae_UCG_003和Tyzzerella可能与靛玉红对UC的治疗作用相关。结论靛玉红能够通过调节UC小鼠肠道菌群、抑制肠道炎性反应,从而治疗UC。Objective To investigate the effect and mechanism of indirubin on intestinal flora of mice with ulcerative colitis(UC)induced by dextran sulfate sodium(DSS).Methods Male kunming mice were randomly divided into control group,model group,salazosulfapyridine(125 mg/kg)group,and low-,medium-,high-dose(7.5,15.0,30.0 mg/kg)indirubin groups,UC model was induced by free drinking 3%DSS for 7 d.On 8th day,mice were ig drugs,after continuous intervention for 7 d,signs of mice were observed for disease activity index(DAI)score;Hematoxylin-eosin(HE)staining was used to observe the pathological changes of colon tissue;ELISA method was used to detect levels of interleukin-6(IL-6),IL-8,IL-10,IL-1βand tumor necrosis factor-α(TNF-α)in serum of mice.Five samples of intestinal contents in each group were collected,and bacterial 16S rRNA gene V4—V5 regions high-throughput sequencing were used to analyze the flora of intestinal contents variety.Results Compared with model group,DAI score in indirubin group was significantly reduced(P<0.05),colon tissue damage was reduced,inflammatory cell infiltration was decreased,levels of pro-inflammatory cytokines such as IL-6,IL-8,IL-1βand TNF-αin serum were significantly decreased(P<0.05),level of anti-inflammatory cytokine IL-10 showed an increasing trend.16S rRNA gene sequencing results showed intestinal flora diversity of mice in model group was decreased,and intestinal flora diversity in indirubin group was increased;Ratio of Bacteroides/Firmicutes was increased at phylum level,seven genus such as Lachnospiraceae_FCS020_group,Ruminiclostridium_9,Eubacterium_brachy_group,Allobaculum,Streptococcus,Erysipelotrichaceae_UCG_003 and Tyzzerella may be related to the therapeutic effect of indirubin on UC.Conclusion Indirubin can treat UC by regulating intestinal flora of UC mice and inhibiting intestinal inflammation.

关 键 词：靛玉红 溃疡性结肠炎 16S rRNA基因测序 肠道菌群 多样性分析 

分 类 号：R285.5[医药卫生—中药学]